Abstract: The aim of this study was to prepare recombinant epitope protein of Streptococcus suis (S.suis) by constructing prokaryotic expression vector pET28a-DCpep-GSE.The transmembrane structures, signal peptides, B cell epitopes, Th cell epitopes and secondary structures of GAPDH, Enolase and SsnA of S.suis were analyzed and predicted by bioinformatics.The amino acid sequence of the recombinant protein (DCpep-GSE protein) was designed and its theoretical isoelectric point, hydrophilicity and other physicochemical properties were analyzed.Recombinant prokaryotic expression vector pET28a-DCpep-GSE was constructed and transformed into E.coli BL21 (DE3).The induced expression conditions were optimized, and the target protein was purified by His nickel column and tested for cytotoxicity and hemolysis.The results showed that the amino acids at sites 1-50 and 1 036-1 059 of SsnA protein were intracellular or transmembrane segments, and the amino acids at sites 1-56 were signal peptides.GAPDH and Enolase had no transmembrane regions or signal peptides.B and Th cell epitopes were located in random coil and β-turn region of secondary structure, showed that epitopes had good antigenicity.DCpep-GSE protein contained 373 amino acids, with molecular weight of 45.3 ku, theoretical isoelectric point (pI) of 4.57, and GRAVY of -0.677, belonged to the category of acidic hydrophilic protein.A 5 369 bp vector band and a 1 131 bp target band were obtained by double digestion of the plasmid.The sequencing results of PCR amplification products were consistent with the designed sequence, and the vector construction was correct.The highest protein expression was induced by 1 mmol/L IPTG at 37 ℃ for 6 h, and the soluble protein was obtained after ultrasonic crushing.The relative proliferation rate of RAW264.7, PK15 and MARC145 cells were 92.3%, 99.5% and 99.7%, respectively, when the protein concentration was 500 μg/mL in experimental group.The cell morphology was the same as that in control group, and the cells grew well.The hemolysis rate of each sample group was less than 5%.In this study, the recombinant protein DCpep-GSE was designed by bioinformatics method, and then DCpep-GSE was successfully expressed and purified by prokaryotic expression system, and its safety was verified.It laid a foundation for the further development of subunit vaccine of S.suis.

Key words: Streptococcus suis; DC targeting peptide; antigenic epitopes; subunit vaccine; cytotoxicity